IL2 mutants and protein complexes containing them

Abstract

The present invention relates to an IL2 variant and a protein complex containing the same, as well as a method for producing and using the same. The protein complex can selectively increase the activity of effector T cells, thereby reducing side effects and maximizing anti-cancer activity, and can be used to prevent or treat various immune diseases related to cancer.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2022-0105643 filed on August 23, 2022, and Korean Patent Application No. 10-2023-0057361 filed on May 2, 2023, the entire specifications of which are incorporated herein by reference.

[0002] The present invention relates to an IL2 mutant, a protein complex containing the same, a method for producing the same, and uses thereof. [Background technology]

[0003] Immune checkpoints are proteins on the surface of immune cells that cancer cells use to avoid attack by the immune system. Cancer cells express PD-L1 on their cell surface and bind to PD-1, a type of immune checkpoint on T cells, thereby suppressing T cell activation and their ability to kill cancer cells. Immune checkpoint inhibitors activate the human immune system and allow immune cells to selectively attack cancer cells, partially addressing the side effects and resistance issues of existing anticancer drugs that directly attack cancer cells. Numerous immune checkpoint inhibitors have been approved for marketing after demonstrating efficacy in a variety of cancer types. However, their overall response rate (ORR) averages around 30%, leaving many challenges to be overcome. To overcome these challenges, interleukins are gaining importance as a combination or dual-acting agent with immune checkpoint inhibitors to enhance their efficacy.

[0004] Among these, interleukin-2 (IL2) is a 15.5 kDa globular glycoprotein with a length of 133 amino acids that plays a central role in lymphocyte production, survival, and homeostasis. IL2 is primarily synthesized by activated T cells, particularly CD4+ helper T cells, and stimulates T cell proliferation and differentiation, as well as the generation of cytotoxic T lymphocytes (CTLs) and the generation and proliferation of natural killer cells (NK cells). Therefore, IL2 can expand lymphocyte populations and enhance immune cell function in vivo. IL2 therapy is currently approved and used in patients with metastatic renal cell carcinoma and malignant melanoma.

[0005] The activity of IL2 on lymphocytes is mediated by its binding to a combination of three distinct IL2 receptors (IL2Rs), termed IL-2 receptor α (IL2 receptor α, IL2Rα; CD25), IL-2 receptor β (IL2 receptor β, IL2Rβ; CD122), and common cytokine receptor γ (IL2Rγ; CD132). The distribution of each subunit receptor varies from cell to cell, and the binding avidity of IL2 varies significantly among receptors. The high-affinity IL2R consists of a trimer of three subunits (α, β, and γ), while the dimeric IL2 receptor consisting of β and γ subunits is called the intermediate-affinity IL2R. The dimeric intermediate-affinity IL2R binds IL2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, but both the dimeric and trimeric IL2Rs can transmit signals upon IL2 binding. Therefore, the α-subunit, CD25, is not essential for IL2 signaling. While the α-subunit confers high-affinity binding to its receptor, the α- and α-subunits are important for signaling. Trimeric IL2 containing CD25 is expressed by regulatory T cells and endothelial cells. Furthermore, while the trimeric IL2R is transiently induced on normal activated T cells, these T cells express only the dimeric IL2R in the resting state (Nature Review Immunology. 2012 12:180-190).

[0006] Regulatory T cells are a group of T cells that regulate the immune system, maintaining immune tolerance to self-antigens and involved in autoimmune diseases. They generally inhibit the efficacy of cancer treatment by suppressing or downregulating the activation and proliferation of effector T cells. Regulatory T cells persistently express the highest levels of CD25 and have a stronger IL2 binding ability than effector T cells, raising concerns that they may inhibit the efficacy of cancer treatment using IL2.

[0007] Side effects caused by recombinant human IL2 therapy have become a concern with IL2 immunotherapy. Patients receiving high-dose IL2 therapy frequently experience systemic side effects, including severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, and hematological disorders, which require intensive monitoring and hospitalization. The primary cause of these side effects is the development of so-called vesicular leakage syndrome (VLS), a pathological increase in vascular permeability that leads to fluid extravasation in multiple organs (e.g., causing pulmonary and cutaneous edema and hepatocyte damage) and intravascular fluid depletion (causing a drop in blood pressure and a compensatory increase in heart rate). There is no treatment for VLS other than discontinuing IL2 administration. Low-dose IL2 administration has been tested in patients to avoid VLS, but this resulted in inadequate therapeutic effects, resulting in reduced cancer treatment efficacy. VLS was thought to be caused by the release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, from IL2-activated NK cells; however, it has recently been demonstrated that IL2-induced pulmonary edema results from the direct binding of IL2 to pulmonary endothelial cells, which express low or intermediate levels of trimeric IL2R (International Immunology, 2006, vol. 18, no. 10: 1461-1471).

[0008] Therefore, despite the potential of IL2 as an immune anti-cancer therapeutic agent, in order to reduce toxicity and side effects and increase therapeutic efficacy, it is necessary to develop new therapeutic methods that can minimize side effects and increase efficacy of IL2-based cancer treatment by utilizing IL2 variants that selectively activate effector T cells and their associated Fc (fragment crystallizable) regions, immune checkpoint inhibitors, and antibodies against cancer cell-specific antigens. Summary of the Invention [Problem to be solved by the invention]

[0009] One aspect is to provide a protein comprising an IL2 variant.

[0010] Another aspect provides a protein complex comprising an IL2 variant, a first polypeptide comprising a first CH3 antibody constant region and a second polypeptide comprising a second CH3 antibody constant region; and an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor, or an antibody or antigen-binding fragment thereof against a tumor-associated antigen.

[0011] Yet another aspect is to provide a method for producing said protein or said protein complex.

[0012] Yet another aspect is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the protein or the protein complex as an active ingredient.

[0013] Yet another aspect is to provide a method for treating cancer comprising administering said protein or said protein complex.

[0014] Yet another aspect is to provide a use of the protein or protein complex for the manufacture of a cancer therapeutic agent. [Means for solving the problem]

[0015] One aspect provides a protein comprising an IL2 variant, wherein the IL2 variant comprises one or more amino acids selected from the group consisting of glutamic acid (E), alanine (A), lysine (K), and serine (S) at one or more positions selected from the group consisting of 35, 38, 42, and 125.

[0016] The IL2 variant may also be one in which one or more amino acids of wild-type IL2, including the amino acids of SEQ ID NO: 16, have been substituted. The IL2 variant may also be one in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids have been substituted, so long as IL2 activity is maintained. For example, the IL2 variant may also be one in which one or more positions selected from the group consisting of 18, 19, 35, 38, 42, 125, and 126 of wild-type IL2, including the amino acids of SEQ ID NO: 16, have been substituted.

[0017] Specifically, the IL2 variant may comprise one or more amino acids selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), threonine (T), glutamic acid (E), and lysine (K) at one or more positions selected from the group consisting of 18, 19, 35, 38, 42, 125, and 126.

[0018] The IL2 mutants may have increased or decreased binding affinity to IL2 receptors due to amino acid substitutions. For example, the binding affinity to IL2Rα and / or IL2Rβγ may be decreased. Therefore, by decreasing the binding affinity to IL2R, the IL2 mutants may selectively activate effector T cells over regulatory T cells that inhibit IL2-mediated immune activity.

[0019] In one embodiment, the IL2 variant may comprise a glutamic acid (E) at position 35, an alanine (A) at position 38, a lysine (K) at position 42, and a serine (S) at position 125.

[0020] In another embodiment, the IL2 variant may further comprise one or more amino acids selected from the group consisting of leucine (L), methionine (M), arginine (R), alanine (A), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), and threonine (T) at one or more positions selected from the group consisting of 18 and 19.

[0021] In yet another embodiment, the IL2 variant may further comprise one or more amino acids at position 126 selected from the group consisting of threonine (T) and isoleucine (I).

[0022] For example, the IL2 mutant may be an amino acid selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), and glutamine (Q) at position 18; an amino acid selected from the group consisting of serine (S), leucine (L), tryptophan (W), asparagine (N), isoleucine (I), threonine (T), alanine (A), methionine (M), and phenylalanine (F) at position 19; glutamic acid (E) at position 35; alanine (A) at position 38; ricin (K) at 42nd place; Serine (S) at position 125; and At position 126, an amino acid selected from the group consisting of threonine (T) and isoleucine (I) may be included.

[0023] Specifically, the IL2 mutant is glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42 and serine (S) at position 125; methionine (M) at position 18, serine (S) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Alanine (A) at position 18, serine (S) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Arginine (R) at position 18, serine (S) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; methionine (M) at position 18, leucine (L) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Leucine (L) at position 18, serine (S) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Serine (S) at position 18, tryptophan (W) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42 and serine (S) at position 125; Phenylanine (F) at position 18, asparagine (N) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Valine (V) at position 18, isoleucine (I) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Isoleucine (I) at position 18, threonine (T) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42 and serine (S) at position 125; Glutamine (Q) at position 18, alanine (A) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; methionine (M) at position 18, methionine (M) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; methionine (M) at position 18, phenylalanine (F) at position 19, glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125; Glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, serine (S) at position 125, and threonine (T) at position 126; and It may include a residue selected from the group consisting of glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, serine (S) at position 125, and isoleucine (I) at position 126.

[0024] In yet another embodiment, the IL2 variant may comprise an amino acid selected from the group consisting of SEQ ID NOs: 1 to 15. The IL2 variant may also comprise a polynucleotide encoding the amino acid. Specifically, the polynucleotide may be selected from the group consisting of SEQ ID NOs: 56 to 70.

[0025] The protein may also comprise an Fc region attached by a linker or carrier, for example, the linker may comprise 1 to 50 amino acids, albumin or a fragment thereof, or a copolymer such as polyethylene glycol.

[0026] In one embodiment, the linker may comprise the amino acid sequence of SEQ ID NO:18.

[0027] In other specific examples, the Fc region may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19-21; SEQ ID NOs: 22-24; SEQ ID NOs: 25-27; SEQ ID NOs: 28-30; SEQ ID NOs: 31-33; SEQ ID NOs: 34-36; SEQ ID NOs: 37-39; SEQ ID NOs: 40-42; SEQ ID NOs: 43-45; and SEQ ID NOs: 46-48.

[0028] The protein may further comprise an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor, such as PD-L1, PD-1, LAG3, VISTA, BTLA, TIM3, TIGIT, or CTLA-4.

[0029] The protein may further comprise an antibody or antigen-binding fragment thereof against a tumor-associated antigen, such as PD-L1, EGFR, HER-2, B7H3, GPC3, CEA, TROP, or PSMA.

[0030] Thus, another embodiment provides a protein complex comprising an IL2 variant; and an Fc region.

[0031] Yet another embodiment provides a protein complex comprising an IL2 mutant; and an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor.

[0032] Another embodiment provides a protein complex comprising an IL2 variant; and an antibody or antigen-binding fragment thereof against a tumor-associated antigen.

[0033] As used herein, the term "protein complex" refers to a complex of two or more related polypeptides, an artificial recombinant protein expressed by linking the genes of one or more other proteins to a protein, and may be used interchangeably with "fusion protein." The protein complex may be expected to exhibit a synergistic effect in its functions by linking two or more proteins. Therefore, the protein complex may be a conjugate, protein complex, or fusion protein containing an Fc region, which may selectively increase the activity of effector T cells over regulatory T cells by substituting specific amino acids in IL2. Furthermore, the protein complex specifically distributes to the tumor microenvironment, inhibits immune checkpoints, and activates immune cells, thereby effectively inducing the death of cancer cells. This may provide a therapeutic agent with reduced side effects and maximized anticancer activity compared to conventional therapeutic agents.

[0034] Another aspect provides a protein complex comprising a first polypeptide comprising a first CH3 antibody constant region and a second polypeptide comprising a second CH3 antibody constant region, wherein the first and second polypeptides form a heterodimer, and the N-terminus of at least one of the first or second polypeptides comprises an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor, or an antibody or antigen-binding fragment thereof against a tumor-associated antigen, and at least one of the N-terminus or C-terminus of the first or second polypeptide comprises an IL2 variant.

[0035] FIG. 4 shows the structure of a protein complex containing an IL2 mutant according to one embodiment.

[0036] Referring to Figure 4, the protein complex may include an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof against a tumor-specific antigen at the N-terminus of a first polypeptide comprising a first CH3 antibody constant region, and an IL2 variant at the N-terminus of a second polypeptide comprising a second CH3 antibody constant region.

[0037] The protein complex may also comprise an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor, or an antibody or antigen-binding fragment thereof against a tumor-specific antigen, at the N-terminus of a first polypeptide comprising a first CH3 antibody constant region and a second polypeptide comprising a second CH3 antibody constant region, and an IL2 variant at the C-terminus of the first or second polypeptide.

[0038] The specific details of the IL2 variant, immune checkpoint inhibitor, and tumor-specific antigen are as described above. In the protein complex, the binding strength of the antibody or antigen-binding fragment thereof against the immune checkpoint inhibitor to cancer cells or T cells is superior to that of T cells to the IL2 receptor. Therefore, the antibody or antigen-binding fragment thereof against the immune checkpoint inhibitor may primarily bind to cancer cells or T cells surrounding the cancer cells, thereby reducing the side effects caused by IL2 receptor binding, which can induce immune cell activation throughout the body. Furthermore, the antibody or antigen-binding fragment thereof against the tumor-specific antigen not only specifically binds to cancer cells, but also has superior binding strength to cancer cells compared to the binding strength between IL2 receptor-binding proteins. Therefore, the antibody or antigen-binding fragment thereof against the tumor-specific antigen may primarily bind to cancer cells, thereby reducing the side effects caused by IL2 receptor binding, which can induce immune cell activation throughout the body.

[0039] The immune checkpoint inhibitor and / or antibody against a tumor-specific antigen or its antigen-binding fragment may be, for example, an antibody, an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), or a nanobody.

[0040] As used herein, the term "antibody" is used interchangeably with "immunoglobulin (Ig)." A complete antibody has two full-length light chains and two full-length heavy chains, each of which is connected to a heavy chain by a disulfide bond (SS-bond). There are two types of light chains, λ and κ, which consist of approximately 211 to 217 amino acids. Human antibodies each contain only one type of light chain. The light chain consists of a constant region and a variable region. There are five types of heavy chains (γ, δ, α, μ, and ε), and the heavy chain determines the antibody type. α and γ consist of 450 amino acids, while μ and ε consist of 550 amino acids. The heavy chain has two regions: a variable region and a constant region. The variable region refers to the region of an antibody to which an antigen binds. The variable region may contain complementarity determining regions (CDRs) that confer antigen-binding specificity.

[0041] The antibody may comprise an antigen-binding fragment (Fab) region that binds to an antigen and a fragment crystallizable (Fc) region that binds to a cell surface receptor. When cleaved with papain, a complete antibody can be cleaved into two Fab regions and one Fc region. The Fab region may comprise a polypeptide comprising a heavy chain variable region (VH) domain and a heavy chain constant region 1 (CH1) domain, and a polypeptide comprising a light chain variable region (VL) domain and a light chain constant region (CL) domain, linked by disulfide bonds. The Fc region may comprise two polypeptides comprising a heavy chain constant region 2 (CH2) domain and a heavy chain constant region 3 (CH3) domain, linked together. The Fc region may form a hinge region. The CH3 antibody constant region refers to the heavy chain constant region 3 domain of an antibody.

[0042] Generally, the effector function of an antibody is mediated by the Fc region binding to its receptor, the Fcγ receptor, or the complement component molecule C1q, resulting in antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Because immune cells have numerous Fcγ receptors, which can induce the death of unwanted immune cells, it is desirable to remove the effector function. Therefore, the CH3 antibody constant region may be modified to have reduced Fcγ receptor binding or to have no or significantly reduced effector function, thereby achieving high stability. The CH3 antibody constant region may also be modified to reduce antibody-dependent cellular cytotoxicity (ADCC).

[0043] In one specific example, the Fc region may comprise a tryptophan (W) at position 366 in the first CH3 antibody constant region, a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407 in the second CH3 antibody constant region, and a glycine (G) or phenylalanine (F) at position 351 in the second CH3 antibody constant region.

[0044] The Fc region may also contain serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 in the first CH3 antibody constant region, tryptophan (W) at position 366 in the second CH3 antibody constant region, and glycine (G) or phenylalanine (F) at position 351 in the first CH3 antibody constant region.

[0045] The Fc region may also contain tryptophan (W) at position 366 of the first CH3 antibody constant region, glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 of the second CH3 antibody constant region; and phenylalanine (F) or tryptophan (W) at position 351 of the first CH3 antibody constant region.

[0046] The Fc region may also contain glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407 in the first CH3 antibody constant region, tryptophan (W) at position 366 in the second CH3 antibody constant region, and phenylalanine (F) or tryptophan (W) at position 351 in the second CH3 antibody constant region.

[0047] In another specific example, the first CH antibody constant region and the second CH antibody constant region may further comprise one or more amino acids selected from the group consisting of alanine (A), glycine (G), glutamine (Q), phenylalanine (F), glutamic acid (E), and serine (S) at one or more positions selected from the group consisting of 234, 235, 329, 297, 331, and 265. Specifically, the CH antibody constant region is also a CH2 antibody constant region.

[0048] For example, the first CH2 antibody constant region and the second CH2 antibody constant region may further have leucine (L) at positions 234 and 235 substituted with alanine (A) (L234A / L235A).

[0049] In addition, the first CH2 antibody constant region and the second CH2 antibody constant region also have the leucine (L) at positions 234 and 235 substituted with alanine (A), and the proline (P) at position 329 further substituted with glycine (G) (L234A / L235A / P329G).

[0050] The first CH2 antibody constant region and the second CH2 antibody constant region may also have asparagine (N) at position 297 further substituted with alanine (A), glutamine (Q), or glycine (G) (N297A, N297Q, or N297G).

[0051] In addition, the first CH2 antibody constant region and the second CH2 antibody constant region also have leucine (L) at positions 234 and 235 substituted with phenylalanine (F) and glutamic acid (E), and proline (P) at position 331 further substituted with serine (S) (L234F / L235E / P331S).

[0052] In addition, the first CH2 antibody constant region and the second CH2 antibody constant region also have leucine (L) at positions 234 and 235 substituted with phenylalanine (F) and glutamic acid (E), and aspartic acid (D) at position 265 further substituted with alanine (A) (L234F / L235E / D265A).

[0053] In other specific examples, the Fc region may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19 to 21; SEQ ID NOs: 22 to 24; SEQ ID NOs: 25 to 27; SEQ ID NOs: 28 to 30; SEQ ID NOs: 31 to 33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37 to 39; SEQ ID NOs: 40 to 42; SEQ ID NOs: 43 to 45; and SEQ ID NOs: 46 to 48.

[0054] In another embodiment, the first or second polypeptide and the IL2 variant may be linked by a linker or a carrier. The linker may comprise the amino acid sequence (GGGGS)n, where "n" is a natural number from 1 to 10. Specifically, the linker may comprise the amino acid sequence of SEQ ID NO: 18.

[0055] The first and second polypeptides may form the Fc region of an antibody.

[0056] The heterodimer refers to a combination of two polypeptides that differ in the order, number, or type of amino acid residues. The protein complex is also a protein complex formed by the combination of two polypeptides that specifically bind to different targets.

[0057] The protein complex may be an antibody or an antigen-binding fragment thereof, a receptor-agonist conjugate, a receptor-antagonist conjugate, a receptor-ligand conjugate, or a ligand-decoy receptor conjugate. The protein complex may also include a member selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), an extracellular domain of a membrane receptor, an agonist, an antagonist, a ligand, a decoy receptor, a cytokine, a coagulation factor, and an affinity tag.

[0058] The antibody may be, for example, IgA, IgD, IgE, IgG, or IgM. The antibody may be a monoclonal or polyclonal antibody. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

[0059] As used herein, the term "antigen-binding fragment" refers to a portion of a polypeptide that is a fragment of the entire immunoglobulin structure and contains a portion capable of binding to an antigen. For example, an antigen-binding fragment may be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof.

[0060] As used herein, the term "receptor" refers to a substance that receives or transmits a signal that can be transmitted to a biological system. The receptor may also be a protein receptor. The receptor may bind to an agonist, antagonist, ligand, or cytokine. The agonist is a substance that binds to a receptor to activate it and induce a biological response. The antagonist is a substance that binds to a receptor to inhibit it and suppress a biological response. The ligand is a substance that binds to a receptor. The ligand may bind to a decoy receptor. A decoy receptor is a receptor that specifically binds to a ligand and inhibits actual receptor-mediated signal transmission. A cytokine is a small protein that acts to regulate and maintain cell signal transmission and inflammatory processes.

[0061] The protein complex may be modified. For example, the protein complex may be modified by conjugation, glycosylation, tagging, or a combination thereof. The antibody may be conjugated with other drugs, such as anticancer drugs. For example, the protein complex may be conjugated with horseradish peroxidase (HRP), alkaline phosphatase, a hapten, biotin, streptavidin, a fluorescent substance, a radioactive substance, a quantum dot, polyethylene glycol (PEG), a histidine tag, or a combination thereof. The fluorescent substance may be Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 680, Alexa Fluor 750, Alexa Fluor 790, or Alexa Fluor 350 (all registered trademarks).

[0062] The amino acid positions of the Fc region or the CH2 and CH3 regions are based on the Kabat EU index (EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The amino acid positions and corresponding amino acid types of the CH3 domain are based on human IgG1.

[0063] Another aspect provides a polynucleotide encoding a protein according to one aspect or a protein complex according to another aspect.

[0064] The specific details of the protein or protein complex are as described above.

[0065] In one embodiment, the polynucleotide may comprise one or more polynucleotides selected from the group consisting of SEQ ID NOs: 56-70; and one or more polynucleotides selected from the group consisting of SEQ ID NOs: 74-76.

[0066] In other embodiments, the polynucleotide may comprise one or more polynucleotides selected from the group consisting of SEQ ID NOs: 56-70; a polynucleotide comprising SEQ ID NO: 73; and one or more polynucleotides selected from the group consisting of SEQ ID NOs: 74-76.

[0067] In yet other embodiments, the polynucleotide may comprise one or more polynucleotides selected from the group consisting of SEQ ID NOs: 56-70; a polynucleotide comprising SEQ ID NO: 73; one or more polynucleotides selected from the group consisting of SEQ ID NOs: 74-76; and one or more polynucleotides selected from the group consisting of SEQ ID NOs: 77-82.

[0068] Another embodiment provides a method for producing a protein or protein complex, the method comprising transforming a cell with an expression vector encoding a protein according to one embodiment or a protein complex according to another embodiment, thereby expressing the protein or protein complex. Another embodiment also provides a protein or protein complex comprising an IL2 mutant produced by the method.

[0069] The specific details of the protein or protein complex are as described above.

[0070] An expression vector is an expression vector capable of expressing a target protein in an appropriate host cell, and includes essential regulatory elements operably linked to allow the inserted nucleic acid sequence to be expressed. The term "operably linked" refers to the functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid encoding a target protein so that they can perform their general functions. The expression vector also includes a polynucleotide encoding the protein complex. The expression vector may include regulatory elements necessary for gene expression, such as an enhancer, promoter, poly(A) sequence, etc.

[0071] The cell may also be a cancer cell. The cell may also be an in vitro cell. The cell may also be a bacterium, yeast, plant cell, or mammalian cell. The bacterium may also be Escherichia coli. The mammalian cell refers to a cell derived from a mouse, rat, rabbit, dog, cat, sheep, cow, horse, monkey, chimpanzee, or human. The cell may also be a cell line. The cell may, for example, be selected from the group consisting of Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, baby hamster kidney (BHK) cells, NS0 cells, PER.C6 cells, HeLa cells, MDCK (Madin-Darby Canine Kidney) cells, SP2 / 0 mouse myeloma cells, COS-7, and YB2 / 0 rat myeloma cells. The CHO cells may also be CHO DG44, CHO-K1, CHO-S, GS-CHO or CHO DUKX (DXB11) cells. The HEK cells may also be HEK293 cells.

[0072] "Transformation" refers to the process of inserting a specific nucleic acid fragment into the genome of a cell, resulting in expression of the inserted nucleic acid.

[0073] In one embodiment, the method may include co-transfecting a cell with an expression vector encoding a first and / or second polypeptide comprising an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof against a tumor-associated antigen at the N-terminus of at least one of the first or second polypeptides, and an expression vector encoding the first or second polypeptide comprising an IL2 variant at the N-terminus or C-terminus.The method may also include transforming two or more types of cells with an expression vector encoding the first and / or second polypeptide comprising an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof at the N-terminus, and an expression vector encoding the first or second polypeptide comprising IL2 at the N-terminus or C-terminus, respectively.

[0074] The cells may be cultured in a cell culture medium. Cell culture medium refers to a solution containing nutrients necessary for culturing cells. The medium includes commercial or manufactured media used to culture cells. The cell culture medium may include antibiotics. The cell culture medium may include G418 (geneticin), puromycin, blasticidin, zeocin, or a combination thereof. The cell culture medium may include a chemically defined medium.

[0075] The cells may be cultured under conditions that allow cell survival or proliferation. The conditions that allow cell survival or proliferation may vary depending on the cell type. The cells may be cultured at about 25°C to about 42°C, about 25°C to about 40°C, about 30°C to about 40°C, about 30°C to about 37°C, or about 37°C. The cells may be cultured in the presence of air containing about 1% CO2 to about 10% CO2, or about 5% CO2 to about 10% CO2. The cells may be cultured in a medium at about pH 6 to about pH 8, about pH 6.2 to about pH 7.8, about pH 6.4 to about pH 7.6, about pH 6.6 to about pH 7.4, or about pH 6.8 to about pH 7.2. The cells may be cultured under conditions of dissolved oxygen of about 10% to about 80%, about 15% to about 70%, or about 20% to about 60%.

[0076] The culturing method may vary depending on the type of cells. Well-known methods can be used for the culturing. The culturing may be performed on plates, flasks, etc. The culturing may be performed by attaching the cells to a substrate or by suspending the cells in a culture medium. The culturing may be subculture, batch culture, fed-batch culture, perfusion culture, or a combination thereof. During the culturing, the cell culture medium may be periodically replaced with fresh medium. The cells can be cultured for about 1 day or more, about 2 days or more, about 3 days or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 10 days or more, about 2 weeks or more, about 3 weeks or more, about 1 month or more, about 1 day to about 1 month, about 1 day to about 3 weeks, about 1 day to about 2 weeks, about 2 days to about 2 weeks, about 3 days to about 2 weeks, about 4 days to about 2 weeks, about 5 days to about 2 weeks, about 6 days to about 2 weeks, or about 1 week to about 2 weeks.

[0077] The method may further include the step of harvesting the protein complex from the cells or cell culture medium.

[0078] The cell culture medium is also a culture medium free of the cells.

[0079] When the expression vectors are co-transfected into cells, a protein complex of a first polypeptide comprising an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof against a tumor-associated antigen at its N-terminus and a second polypeptide comprising an IL2 variant at its N-terminus or C-terminus can be obtained from the cells or cell culture medium.When the expression vector encoding the first polypeptide comprising an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof against a tumor-associated antigen at its N-terminus and the expression vector encoding the second polypeptide comprising an IL2 variant at its N-terminus or C-terminus can be respectively obtained from the cells or cell culture medium.

[0080] The step of obtaining the protein complex may include incubating the obtained first polypeptide comprising, at its N-terminus, an antibody or antigen-binding fragment thereof against an immune checkpoint inhibitor or an antibody or antigen-binding fragment thereof against a tumor-associated antigen with the obtained second polypeptide comprising an IL2 variant at its N-terminus or C-terminus to form a protein complex. The incubation may be performed under reducing conditions, such as in the presence of 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or a combination thereof.

[0081] The step of obtaining the protein complex may include purifying the protein complex, which may be accomplished by filtration, centrifugation, chromatography, dialysis, immunoprecipitation, or a combination thereof.

[0082] Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising a protein according to one aspect or a protein complex according to another aspect. Yet another aspect provides use of a protein according to one aspect or a protein complex according to another aspect for the manufacture of a medicament for preventing or treating cancer.

[0083] The specific details regarding the protein or the protein complex are as described above.

[0084] The cancer may be a solid cancer or a non-solid cancer. A solid cancer refers to a cancer tumor that develops in an organ, such as the liver, lung, breast, or skin. A non-solid cancer is a cancer that develops in the blood and is also called a blood cancer. The cancer may be a carcinoma, sarcoma, hematopoietic cell-derived cancer, germ cell tumor, or blastoma. The cancer may be selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, late-developing leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

[0085] The term "prevention" refers to any action that suppresses or delays the onset of a disease by administering the pharmaceutical composition. The term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering the pharmaceutical composition.

[0086] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "carrier" is used to include excipients, diluents, or adjuvants. The carrier may be, for example, selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, saline, buffers such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may also include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

[0087] The pharmaceutical composition can be prepared in any dosage form by a conventional method. The composition can be formulated, for example, into an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection). The composition can also be prepared into a systemic dosage form or a local dosage form.

[0088] The pharmaceutical composition may further contain another anti-cancer drug. The anti-cancer drug may be cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, Perjeta, lapatinib, paclitaxel, taxol, tamoxifen, cisplatin, or a combination thereof. The pharmaceutical composition may be a single composition or separate compositions. For example, the composition of the antibody or antigen-binding fragment thereof may be a parenteral dosage form, and the anti-cancer drug may be an oral dosage form.

[0089] The pharmaceutical composition may contain an effective amount of the protein complex. The term "effective amount" refers to an amount sufficient to exhibit a preventive or therapeutic effect when administered to an individual in need of disease prevention or treatment. The effective amount can be appropriately selected by those skilled in the art depending on the cell or individual selected. It can be determined based on factors such as the severity of the disease, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the administration time, administration route and excretion rate, the duration of treatment, drugs used in combination with or coadministered with the composition, and other factors well known in the medical field. The effective amount may be about 0.5 μg to about 2 g, about 1 μg to about 1 g, about 10 μg to about 500 mg, about 100 μg to about 100 mg, or about 1 mg to about 50 mg per pharmaceutical composition.

[0090] The dosage of the pharmaceutical composition is, for example, within the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg for an adult. The administration can be once a day, multiple times a day, once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0091] Another embodiment provides a method for preventing or treating cancer comprising administering to a cell or an individual a protein according to one embodiment or a protein complex according to another embodiment.

[0092] Specific details regarding the protein, protein complex, cell, cancer, prevention, or treatment are as described above.

[0093] The individual may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The individual may also be an individual suffering from or at risk of suffering from cancer.

[0094] The method may further comprise administering to the individual a second active ingredient, which is also an active ingredient for preventing or treating cancer. The second active ingredient may be administered simultaneously with, separately from, or sequentially to the protein complex.

[0095] The protein or protein complex may be administered directly to an individual by any means, such as, for example, oral, intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The protein complex may be administered systemically or locally, and may be administered alone or in combination with other pharmaceutically active compounds.

[0096] The desired dosage of the protein or protein complex varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art. The dosage may be, for example, within the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg for an adult. The dosage may be once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year. [Effects of the Invention]

[0097] The protein according to one embodiment can selectively increase the activity of effector T cells over regulatory T cells by replacing a specific amino acid sequence in IL2. Furthermore, pharmaceutical compositions containing the protein can have reduced side effects compared to conventional therapeutic agents and maximize anti-cancer activity, and can be used for the prevention or treatment of cancer. [Brief explanation of the drawings]

[0098] [Figure 1A] Figure 1 shows the purification of an embodiment of a PD-L1 antibody-IL2 variant 2 protein complex using a Protein-A affinity column purification method. [Figure 1B] Figure 1 shows the purification of an embodiment of a PD-L1 antibody-IL2 variant 2 protein complex using a cation exchange resin column purification method. [Figure 1C] Figure 1 shows the purity analysis of a PD-L1 antibody-IL2 variant 2 protein conjugate according to one embodiment using SE-HPLC analysis. [Figure 2A] Figure 1 shows the results of comparing the anti-cancer effects of a PD-L1 antibody-IL2 variant 2 protein conjugate according to one embodiment, a positive control group (Avelumab, and a combination of Avelumab and Aldesleukin), and a negative control group in mice bearing colon cancer. [Figure 2B] The results show that the PD-L1 antibody-IL2 mutant 2 protein complex according to one embodiment has the ability to inhibit individual cancer cell growth and perform complete remission in mice implanted with colon cancer. [Figure 2C] The results show that the negative control group and the positive control group (administered Avelumab and a combination of Avelumab and Aldesleukin) were able to confirm the individual cancer cell growth inhibition and complete remission effects in colon cancer-implanted mice. [Figure 3] This shows the results of confirming the anti-cancer effect of a specific example of a PD-L1 antibody-IL2 variant 4 protein complex in mice implanted with colon cancer. [Figure 4] 1 shows the structure of a protein complex containing an IL2 mutant according to one embodiment. [Figure 5A] 1 shows the results of purifying a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a Protein-A affinity column purification method. [Figure 5B] 1 shows the results of purifying a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a hydrophobic interaction column purification method. [Figure 5C] 1 shows the results of purity analysis of a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment. [Figure 6A] 1 shows the results of comparing the anti-cancer effects of a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment, a negative control group, and a positive control group in a mouse model xenografted with colon cancer. [Figure 6B] 1 shows the results of comparing the weight changes of a mouse model implanted with colon cancer after administering a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one specific example, a negative control group, and a positive control group to the mouse model implanted with colon cancer. DETAILED DESCRIPTION OF THE INVENTION

[0099] Hereinafter, preferred examples will be presented to aid in understanding the present invention. However, the following examples are provided to facilitate a more easily understood understanding of the present invention, and the content of the present invention is not limited to the following examples. [Example]

[0100] Example 1. Preparation of IL2 mutant proteins and confirmation of their activity

[0101] 1-1. Cloning and cultivation of IL2 mutant proteins

[0102] IL2 mutants were prepared by substituting amino acids at certain positions in the wild-type IL2 amino acid sequence (SEQ ID NO: 16). Specifically, polynucleotides encoding IL2 mutants tagged with 6XHis were synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific, and then inserted into the ApaII-NheI enzyme site of the pcDNA3.1 expression vector to prepare expression vectors expressing the IL2 mutants. The amino acid substitution sites and substituted amino acids in the IL2 mutants are shown in Table 1 below. Hereinafter, the term "ExpiFectamine" is used. TM The expression vector was transfected into ExpiCHO-S using a CHO Transfection kit (ThermoFisher). TM The cells were transformed into cell lines. The transformed cells were cultured at 32°C in a 5% CO2 incubator at 120 rpm for 12 days. After 12 days, the culture supernatant was separated and collected, and filtered through a sterile filter to obtain the IL2 mutant protein.

[0103] [Table 1]

[0104] 1-2. Purification of IL2 mutant proteins

[0105] The IL-2 mutant protein obtained from the culture medium obtained in Example 1-1 was purified using His tagged protein, and a highly purified material with a purity of 95% or more was obtained.

[0106] First, the culture medium obtained in Example 1-1 was centrifuged to separate the cultured cells from the medium. Then, the IL2 mutant protein in the separated medium was filtered through a 0.22 μm filter (Thermo Scientific) to remove fine residues. The filtered medium was first purified using imidazole affinity chromatography (Ni-SepFast, Biotolomics), followed by desalting and concentration to remove imidazole buffer components present in the purified product. The content and purity of the final purified product were then analyzed. Specifically, the filtered medium was loaded onto a Ni-SepFast column stabilized in phosphate buffer saline (pH 7.4). Nonspecifically bound proteins were washed off using the same buffer, and proteins specifically bound to the Ni-SepFast column were eluted using an imidazole buffer solution (0 M and 0.5 M, pH 7.4) in an imidazole concentration-increasing gradient manner. The isolated IL2 mutant protein was desalted in phosphate buffer saline (pH 7.4) and stabilized. The purity of the purified IL2 mutant protein was analyzed using size-exclusion HPLC (TSK-3000SWxL, 7.8 mm x 30 cm, Tosoh Co.), and the molecular weight was confirmed by SDS-PAGE analysis.

[0107] 1-3. Confirmation of activation of IL2 mutant proteins on human immune cells

[0108] The activity of the purified IL2 variant-protein complexes in human immune cells was confirmed. Specifically, human peripheral blood mononuclear cells (Stemcell) were reacted with antibodies specifically fluorescently labeled to effector CD8+ T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) for 30 minutes at 4°C in the dark, and then the antibodies not attached to the cells were removed by centrifugation. The IL2 variants 2, 4, and 14 proteins purified in Example 1-2 were then treated and reacted for 20 minutes at 37°C in the dark, and then fixed in 1 mL of fixation buffer (BD, US) for 12 minutes. Aldesleukin (Proleukin, Novartis, Switzerland) was used as a positive control, with each drug administered at concentrations ranging from 0.01 to 8,000 nM. Next, the cytoplasm of fixed human peripheral blood mononuclear cells was treated with 1.5 mL of Perm3 buffer (BD, US) and incubated for 35 minutes at 4°C to allow the fluorescent label to penetrate into the cytoplasm. Afterwards, phosphorylated STAT-5 and Foxp3+ protein, a Treg cell marker, were fluorescently labeled and incubated for 30 minutes. The level of STAT-5 phosphorylation in effector CD8+ T and Treg cells treated with IL2 mutant proteins was compared using flow cytometry.

[0109] [Table 2]

[0110] As a result, as shown in Table 2, the positive control group (Aldesleukin) significantly increased the EC of CD8+ T cells / regulatory T cells. 50 The ratio of these values ​​was 16.3, confirming that the activity of Treg cells was more strongly induced than that of CD8+ T cells.

[0111] On the other hand, IL2 variants 2, 4, and 14 proteins inhibited EC of CD8+ T cells / regulatory T cells. 50The ratios of these values ​​were 0.6, 0.56, and 5.6, respectively, confirming that the activity of CD8+ T cells was more selectively activated compared to the positive control group.

[0112] Example 2. Preparation and characterization of IL2 mutant-Fc protein conjugates

[0113] 2-1. Cloning and culture of IL2 mutant-Fc protein complexes

[0114] Fc protein complexes containing IL2 variants were prepared. Specifically, polynucleotides encoding the Fc region were synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific, and then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector to prepare a first expression vector expressing the Fc region. Using the same service, polynucleotides encoding complexes containing IL2 variants 1, 2, 4, and 14 prepared in Example 1, a linker, and the Fc region were synthesized, and then inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector to prepare a second expression vector expressing IL2 variants-Fc. Subsequently, the polynucleotides were purified using ExpiFectamine. TM The first and second expression vectors were mixed at a ratio of 1:1 using a CHO Transfection kit (ThermoFisher), and then transfected into ExpiCHO-S TM The cells were transformed into a cell line. The transformed cells were cultured at 125 rpm in an 8% CO2 incubator at 32°C for 12 days. After 12 days, the culture supernatant was separated and collected, and filtered through a sterile filter to obtain protein complexes containing Fc and IL2 variant-Fc.

[0115] 2-2. Purification of IL2 mutant-Fc protein complexes

[0116] The IL2 mutant-Fc protein complex obtained through the culture of the cell line prepared in Example 2-1 was purified to obtain a highly pure substance.

[0117] First, the culture medium obtained in Example 2-1 was centrifuged to separate the cultured cells from the medium. Then, the IL2 variant-Fc protein complex in the separated medium was filtered through a 0.22 μm filter (Thermo Scientific) to remove fine debris. The filtered medium was then purified using Protein A affinity chromatography (MabSelect PrismA, Cytiva). Specifically, the filtered medium was loaded onto a Protein-A column stabilized with phosphate buffer saline (pH 7.4), and nonspecifically bound proteins were washed off using the same buffer. Proteins specifically bound to the Protein-A column were eluted using a pH gradient of two solutions containing 0.02 M citric acid, pH 5.0 and pH 3.5. The sample was then neutralized with 1 M Tris at pH 7.2. The obtained IL2 mutant-Fc protein complex was then stabilized in phosphate buffer saline (pH 7.4), and the purity of the purified IL2 mutant-Fc protein complex was analyzed using a size exclusion HPLC (TSK-3000SWxL, 7.8 mm x 30 cm, Tosoh).

[0118] 2-3. Confirmation of activation of IL2 mutant-Fc-protein complexes on human immune cells

[0119] The activity of the purified IL2 variant-Fc protein complexes in human immune cells was confirmed. Specifically, human peripheral blood mononuclear cells (Stem Cell) were incubated with antibodies specifically fluorescently labeled to effector CD8+ T cells (CD3+, CD8+) and Treg cells (CD4+, CD25+) for 30 minutes at 4°C in the dark, and then the antibodies not attached to the cells were removed by centrifugation. The purified IL2 variant 1-Fc protein complex, IL2 variant 2-Fc protein complex, IL2 variant 4-Fc protein complex, and IL2 variant 14-Fc protein complexes were incubated for 20 minutes at 37°C in the dark, and then fixed in 1 mL of fixation buffer (BD, US) for 12 minutes. Aldesleukin (Proleukin, Novartis, Switzerland) was used as a positive control, with each drug administered at concentrations ranging from 0.01 to 8,000 nM. Next, the cytoplasm of fixed human peripheral blood mononuclear cells was treated with 1.5 mL of Perm3 buffer (BD, US) and incubated for 35 minutes at 4°C to allow the fluorescent label to penetrate into the cytoplasm. After that, phosphorylated STAT-5 and Foxp3+ protein, a Treg cell marker, were fluorescently labeled and incubated for 30 minutes. The level of STAT-5 phosphorylation in effector CD8+ T cells and Treg cells treated with IL2 mutant-Fc protein complexes was compared using flow cytometry.

[0120] [Table 3]

[0121] As a result, as shown in Table 3, the positive control group (Aldesleukin) significantly increased the EC of CD8+ T cells / regulatory T cells. 50 The ratio of the values ​​was 33.3, which confirmed that the activity of Treg cells was induced more strongly than that of CD8+ T cells.

[0122] On the other hand, the IL2 variant 1-Fc protein complex, the IL2 variant 2-Fc protein complex, the IL2 variant 4-Fc protein complex, and the IL2 variant 14-Fc protein complex inhibited the EC of CD8+ T cells / regulatory T cells. 50 The ratios of the values ​​were 0.79 to 3.2, confirming that the activity of CD8+ T cells was more selectively activated compared to the positive control group.

[0123] That is, it can be seen that the IL2 variant according to one embodiment can retain the property of more selectively activating CD8+ cells than regulatory T cells even in a structure with an added Fc.

[0124] Example 3. Preparation and characterization of PD-L1 antibody-IL2 mutant protein conjugates

[0125] 3-1. Cloning and culture of PD-L1 antibody-IL2 mutant protein complexes

[0126] A protein conjugate containing PD-L1 antibody and an IL2 variant was prepared. Specifically, a polynucleotide encoding the PD-L1 antibody heavy chain variable region-heavy chain constant region and a polynucleotide encoding the PD-L1 antibody light chain constant region-light chain variable region were synthesized using the same service as in Example 2-1. The polynucleotide encoding the PD-L1 antibody heavy chain variable region-heavy chain constant region and the polynucleotide encoding the PD-L1 antibody light chain constant region-light chain variable region were then inserted into the AvrII-BstZ17I enzyme site and the EcoRV-PacI enzyme site of the pCHO1.0 expression vector, respectively, to prepare a first expression vector expressing PD-L1 antibody. Using the same service, a polynucleotide encoding a conjugate comprising any one of IL2 variants 1 to 15 prepared in Example 1, a linker, and an Fc domain was synthesized and inserted into the AvrII-BstZ17I enzyme site of the pCHO1.0 expression vector to prepare a second expression vector expressing the IL2 variant-Fc. Thereafter, a PD-L1 antibody-IL2 variant protein conjugate comprising anti-PD-L1 Fab-Fc and IL2 variant-Fc was obtained in the same manner as in Example 2-1, except that the first and second expression vectors were used.

[0127] 3-2. Purification of PD-L1 antibody-IL2 mutant protein complex

[0128] The PD-L1 antibody-IL2 variant 1 protein complex or PD-L1 antibody-IL2 variant 15 protein complex obtained through the culture of the cell line prepared in Example 3-1 was purified to obtain a highly pure material.

[0129] First, the culture medium obtained in Example 3-1 was centrifuged to separate the cultured cells from the medium. The PD-L1 antibody-IL2 variant protein complex in the separated medium was then filtered through a 0.22 μm filter (Thermo Scientific) to remove fine debris. The filtered medium was then purified using Protein A affinity chromatography (MabSelect PrismA, Cytiva). Specifically, the filtered medium was applied to a Protein A column stabilized with phosphate buffer saline (pH 7.4), and non-specifically bound proteins were washed off using the same buffer. Proteins specifically bound to the Protein A column were eluted using a buffer solution containing 0.05 M Citric acid (pH 3.9), and the sample was neutralized with 1 M Tris at pH 7.2. Subsequently, to remove impurities derived from substances remaining in the sample separated through the affinity column, a second purification step was performed using cation exchange chromatography (Source30S, Cytiva). Specifically, the sample eluted and neutralized from the Protein-A column was titrated to pH 6.0 by adding 1 M Citric acid, and then loaded onto a Source 30S column stabilized with 20 mM sodium phosphate (pH 6.0) buffer. Non-specifically bound proteins were washed away using the same buffer, and the PD-L1 antibody-IL2 mutant protein complex was isolated by elution using an increasing gradient of a buffer solution containing 0.3 M NaCl (pH 6.0). The isolated PD-L1 antibody-IL2 mutant protein complex was then stabilized with phosphate buffer saline (pH 7.4), and the purity of the purified PD-L1 antibody-IL2 mutant protein complex was analyzed using a size-exclusion HPLC (TSK-3000SWxL, 7.8 mm x 30 cm, Tosoh).

[0130] Figure 1A shows the purification of an embodiment of a PD-L1 antibody-IL2 variant 2 protein complex using a Protein-A affinity column purification method.

[0131] Figure 1B shows the purification of an embodiment of a PD-L1 antibody-IL2 variant 2 protein complex using a cation exchange column purification method.

[0132] Figure 1C shows the purity analysis of a PD-L1 antibody-IL2 variant 2 protein conjugate according to one embodiment using SE-HPLC analysis.

[0133] As a result, as shown in Figure 1A, specific binding of proteins to the Protein-A affinity column was confirmed depending on the elution buffer used in Protein-A affinity column purification. Specifically, PD-L1 antibody-IL2 mutant protein complexes that specifically bound to the column were confirmed in 1500-1550 ml of eluate.

[0134] In addition, as shown in Figure 1B, it was confirmed that the remaining impurities and nonspecifically binding proteins in the eluate used in the Protein-A affinity column were removed.

[0135] Furthermore, as shown in Figure 1C, the PD-L1 antibody-IL2 variant 2 protein complex obtained through Protein-A affinity column purification and cation exchange resin purification exhibited a purity of 99% with a retention time of 16.717 minutes.

[0136] That is, it was found that the PD-L1 antibody-IL2 variant protein according to one embodiment can be purified to a high degree of purity.

[0137] 3-3. Confirmation of receptor binding ability of PD-L1 antibody-IL2 mutant protein complex

[0138] The receptor binding strength of the PD-L1 antibody-IL2 variant protein conjugates according to one embodiment was confirmed by SPR (Surface Plasmon Resonance) analysis. Specifically, human PD-L1 and human IL2Rα, IL2Rβγ, and IL2Rαβγ were each covalently immobilized on a CM5 sensor chip. The PD-L1 antibody-IL2 variant 2 protein conjugate and the PD-L1 antibody-IL2 variant 4 protein conjugate prepared in Example 3-2 were then applied at various concentrations (2-fold serial dilutions in the range of 0.391 to 400 nM) to confirm the binding kinetics for human PD-L1 and IL2 receptor (IL2R), respectively. The binding affinity (KD) was then calculated using the measured association constant (Ka) and dissociation constant (Kd). SPR sensogram analysis was performed using BIAlogue kinetics evaluation software. Avelumab and Aldesleukin were used as positive controls, and the results are shown in Tables 4 and 5 below.

[0139] [Table 4]

[0140] [Table 5]

[0141] Figure 2A shows the binding affinity of a PD-L1 antibody-IL2 mutant 2 protein complex to human PD-L1 antigen according to one embodiment.

[0142] Figure 2B shows the results of confirming the binding ability of Avelumab to the human PD-L1 antigen.

[0143] As a result, as shown in Figure 2A and Table 4, the PD-L1 antibody-IL2 variant 2 protein conjugate prepared in Example 3-2 and the positive control group were confirmed to bind to the human PD-L1 antigen. Specifically, the protein variant showed a binding affinity of 0.48 nM, while the positive control group showed a binding affinity of 0.12 nM, which is approximately 4.1 times lower.

[0144] That is, the positive control group contains a bivalent anti-PD-L1 arm as a single antibody, while the PD-L1 antibody-IL2 variant 2 protein complex contains a monovalent anti-PD-L1 arm, which is thought to result in a difference in binding strength.

[0145] Furthermore, as shown in Table 5, the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 2 bound to IL2Rβγ and IL2Rαβγ with binding affinities of 37.9 nM and 17.3 nM, respectively, but did not react with IL2Rα. Furthermore, the PD-L1 antibody-IL2 variant 2 protein complex bound to IL2Rβγ and IL2Rαβγ with binding affinities of 23.8 nM and 30.6 nM, respectively, but did not react with IL2Rα.

[0146] That is, it was confirmed that the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex according to one embodiment do not bind to IL2 receptor α.

[0147] On the other hand, as shown in Table 5, the positive control group was confirmed to bind to IL2Rα, IL2Rβγ, and IL2Rαβγ with binding affinities of 35.7 nM, 1.55 nM, and 0.10 nM, respectively.

[0148] Meanwhile, the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex were found to have approximately 24.5-fold and 15.4-fold reduced binding avidity to IL2Rβγ, respectively, compared to the positive control. Furthermore, the βγ / αβγ ratio was calculated based on the IL2Rαβγ binding avidity results. The βγ / αβγ ratios for the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex were 2.19 and 0.78, respectively, while the positive control group's ratio was 15.5, confirming that the IL2Rβγ / αβγ ratio for the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex was significantly lower than that of the positive control. This indicates that the PD-L1 antibody-IL2 variant 2 protein complex and the PD-L1 antibody-IL2 variant 4 protein complex not only did not bind to IL2Rα, but also weakened their binding to IL2Rβγ.

[0149] Therefore, in one embodiment, the PD-L1 antibody-IL2 variant protein conjugate is intended to induce higher activation in CD8+ T cells compared to regulatory T cells, and is characterized in that its binding ability to IL2Rα is abolished and its binding ability to IL2Rβγ is regulated through the substitution of the specific amino acids, in order to selectively induce activation of CD8+ T cells that express IL2Rβγ.

[0150] 3-4. Confirmation of the activity of the PD-L1 antibody-IL2 mutant protein complex against human immune cells

[0151] The effect of one embodiment of the PD-L1 antibody-IL2 variant 1 protein conjugate or PD-L1 antibody-IL2 variant 15 protein conjugate on the activation of human immune cells was examined. Specifically, human peripheral blood mononuclear cells (Stem Cell) were reacted with specific fluorescently labeled antibodies against CD8+ T cells (CD3+, CD8+) and regulatory T cells (CD4+, CD25+, FoxP3+), respectively, at 4°C for 30 minutes in the dark. After removing the antibodies not attached to the cells by centrifugation, the human peripheral blood mononuclear cells were treated with the PD-L1 antibody-IL2 variant 1 protein conjugate or PD-L1 antibody-IL2 variant 15 protein conjugate prepared in Example 3-2 at various concentrations (0.001-8000 nM), and reacted at 37°C for 20 minutes in the dark. The cells were then fixed in 1 mL of fixation buffer (BD, US) for 12 minutes. To fluorescently label phosphorylated STAT-5 in the cytoplasm, cells were treated with 1.5 mL of Perm3 buffer (BD, US) and incubated at 4°C for 40 minutes to allow the fluorescent label to permeate. The phosphorylated STAT-5 protein was then fluorescently labeled and incubated for 30 minutes, after which the percentage of cells with phosphorylated STAT-5 was determined by FACS analysis in effector CD8+ T cells and regulatory T cells. Aldesleukin (Proleukin, Novartis, Switzerland) was used as a positive control, and the results are shown in Table 6 below.

[0152] [Table 6]

[0153] *NS: Not saturated at high concentration

[0154] As a result, as shown in Table 6, the EC of CD8+ T cells / regulatory T cells in the positive control group and the PD-L1 antibody-IL2 comparison group 50The ratios of these values ​​were 62.9 and 22, respectively, which indicates the EC of CD8+ T cells / regulatory T cells of the PD-L1 antibody-IL2 variant 1 protein complex and the PD-L1 antibody-IL2 variant 15 protein complex prepared in Example 3-2. 50 The ratios of these values ​​ranged from 0.16 to 2.88. In other words, while the positive control group and the PD-L1 antibody-IL2 comparison group strongly activated regulatory T cells, the PD-L1 antibody-IL2 variant 1 protein complex and the PD-L1 antibody-IL2 variant 15 protein complex prepared in Example 3-2 selectively activated effector T cells, CD8+ T cells, over regulatory T cells.

[0155] Therefore, the PD-L1 antibody-IL2 variant protein complex according to one embodiment may be involved in immune responses related to anti-cancer effects by inducing effector T cell activity more strongly than regulatory T cell activity.

[0156] 3-5. Confirmation of anti-cancer activity of PD-L1 antibody-IL2 mutant protein complex (1)

[0157] The anti-cancer activity of the PD-L1 antibody-IL2 mutant protein conjugate according to one embodiment was confirmed. Specifically, MC38 cells (1×10 6 A syngeneic mouse model of colon cancer was prepared by subcutaneously injecting 0.2 mL / mouse of PD-L1 antibody-IL2 variant 2 protein conjugates prepared in Example 3-2 at doses of 8 mg / kg and 16 mg / kg Q2D x 2 times. After conjugate administration, tumor volume was measured (3 times / week) and anti-cancer activity was compared. As a positive control, avelumab (Merck, Germany) was intraperitoneally administered at a dose of 10 mg / kg Q2D x 2 times, and avelumab and aldesleukin were co-administered (aldesleukin 0.46 mg / kg Q2D x 5 times, ip + avelumab 10 mg / kg, ip, Days 5 and 7).

[0158] Figure 2A shows the results of a comparison of the anti-cancer activity of a PD-L1 antibody-IL2 variant 2 protein conjugate according to one embodiment and a positive control.

[0159] Figure 2B shows the individual cancer cell growth inhibitory effect and complete remission activity of the PD-L1 antibody-IL2 variant 2 protein conjugate according to one embodiment in mice implanted with colon cancer.

[0160] FIG. 2C shows the results of confirming the cancer cell growth inhibitory effect and complete remission activity in the negative control group and the positive control group (administration of Avelumab and a combination of Avelumab and Aldesleukin) in colon cancer-injected mice.

[0161] As a result, as shown in Figure 2A, while tumor volume increased over time in the negative control group (vehicle) and positive control group after administration, the PD-L1 antibody-IL2 variant 2 protein complex prepared in Example 3-2 demonstrated a strong cancer cell growth inhibitory effect. Specifically, the PD-L1 antibody-IL2 variant 2 protein complex was observed to almost completely eliminate tumors at a dose of 16 mg / kg. Furthermore, unlike the PD-L1 antibody-IL2 comparison protein complex, the PD-L1 antibody-IL2 variant 2 protein complex did not result in weight loss or death, regardless of the administered dose.

[0162] Furthermore, as shown in Figures 2B and 2C, tumor volume increased over time after administration in the negative control and positive control groups, and the number of mice showing a complete response (CR) was relatively low, at 0-2 out of 10 experimental animals in the same administration group. In contrast, in the case of the PD-L1 antibody-IL2 variant 2 protein complex, the number of mice showing a complete response increased in a dose-dependent manner, at 6-9 out of 10 experimental animals in the same administration group, demonstrating significant anti-cancer activity compared to the negative control and positive control groups.

[0163] That is, the PD-L1 antibody-IL2 variant protein conjugate according to one embodiment exhibits superior anti-cancer activity compared to the existing antibody therapeutic agents Avelumab and / or Avelumab in combination with Aldesleukin.

[0164] 3-6. Confirmation of anti-cancer activity of PD-L1 antibody-IL2 mutant protein complex (2)

[0165] The anti-cancer activity of one embodiment of the PD-L1 antibody-IL2 variant protein conjugate was confirmed using the PD-L1 antibody-IL2 variant 4 protein conjugate in the same manner as in Examples 3-5 above, except that Avelumab (Merck, Germany) was administered intraperitoneally at a dose of 10 mg / kg (Q2D x 2) as a positive control.

[0166] Figure 3 shows the anti-cancer activity of a PD-L1 antibody-IL2 variant 4 protein complex according to one embodiment.

[0167] As a result, as shown in Figure 3, the negative control group (vehicle) showed an increase in tumor volume over time after administration, whereas the PD-L1 antibody-IL2 variant 4 protein complex prepared in Example 3-2 exhibited a stronger cancer cell growth inhibitory effect compared to the negative control group. Specifically, the PD-L1 antibody-IL2 variant 4 protein complex showed significantly less change in tumor volume at doses of 8 mg / kg and 16 mg / kg compared to the positive control group at a dose of 10 mg / kg. Furthermore, while only one mouse out of ten experimental animals in the positive control group showed complete remission, the number of mice treated with the PD-L1 antibody-IL2 variant 4 protein complex increased to four to five out of ten experimental animals in the same group, demonstrating significant anti-cancer activity compared to the positive control group.

[0168] That is, the PD-L1 antibody-IL2 variant protein conjugate according to one embodiment selectively increases the activity of effector T cells over regulatory T cells compared to existing anti-cancer antibody therapeutics, and may be useful in the prevention or treatment of various immune diseases, including cancer.

[0169] Therefore, the PD-L1 antibody-IL2 mutant protein complex according to one embodiment has excellent anti-cancer activity and reduces the side effects caused by IL2, thereby providing a safer therapeutic agent compared to existing immunological anti-cancer therapeutic agents.

[0170] Example 4. Preparation of mouse PD-1 antibody-IL2 mutant protein complex (a+b) and confirmation of activity

[0171] 4-1. Cloning and cultivation of mouse PD-1 antibody-IL2 mutant protein complex (a+b)

[0172] A protein complex (a+b) containing anti-mouse PD-1 Fab-Fc (hereinafter referred to as "a") and IL2 mutant-Fc (hereinafter referred to as "b") was obtained in the same manner as in Example 3-1, except that the PD-1 antibody and IL2 mutants 1 and 4 prepared in Example 1 were used (see Figure 4A).

[0173] 4-2. Purification of mouse PD-1 antibody-IL2 mutant protein complex (a+b)

[0174] The mouse PD-1 antibody-IL2 mutant protein complex (a+b) obtained through the culture of the cell line prepared in Example 4-1 was purified in the same manner as in Example 3-2 to obtain a highly pure substance.

[0175] 4-3. Confirmation of activity of mouse PD-1 antibody-IL2 mutant protein complex (a+b) on human immune cells

[0176] To confirm the effect of the mouse PD-1 antibody-IL2 mutant protein complex (a+b) obtained in Example 4-2 on the activation of human immune cells, an experiment was carried out in the same manner as in Examples 3-4.

[0177] [Table 7]

[0178] As a result, as shown in Table 7, the CD8+ T cell / regulatory T cell ratios for the positive control group and the mouse PD-1 antibody-IL2 variant 4 protein complex (a+b) were 14.5 and 0.92, respectively. That is, the positive control group strongly activated regulatory T cells, while the mouse PD-1 antibody-IL2 variant 4 protein complex (a+b) prepared in Example 4-2 selectively activated CD8+ T cells, which are effector T cells, over regulatory T cells.

[0179] Therefore, it can be seen that the IL2 mutant protein according to one embodiment retains its original properties well when complexed with not only PD-L1 antibodies but also other immune checkpoint inhibitors, and can participate in immune responses by promoting the activity of effector T cells.

[0180] Example 5. Preparation of mouse PD-1 antibody-IL2 mutant protein conjugate (a+c) and confirmation of activity

[0181] 5-1. Cloning and cultivation of mouse PD-1 antibody-IL2 mutant protein complex (a+c)

[0182] Protein complexes containing mouse PD-1 antibody and IL2 variants 1, 2, 3, 4, 7, 8, 14, and 15 were prepared. Specifically, a polynucleotide encoding the mouse PD-1 antibody heavy chain variable region-constant region and a polynucleotide encoding the mouse PD-1 antibody light chain constant region-light chain variable region were synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific. Each polynucleotide was then inserted into the AvrII-Bstz17I enzyme site and the EcoRV-PacI enzyme site of the pCHO 1.0 expression vector, respectively, to prepare a first expression vector expressing mouse PD-1 antibody. Using the same service, a polynucleotide encoding a conjugate containing any one of IL2 variants 1, 2, 3, 4, 7, 8, 14, and 15 from Example 1 and a linker at the C-terminus of a polynucleotide encoding the mouse PD-1 antibody heavy chain variable region-constant region was synthesized and inserted into the AvrII-BstZ17I enzyme site of the pCHO 1.0 expression vector, and the mouse PD-1 antibody light chain constant region-light chain variable region was inserted into the EcoRV-PacI enzyme site to prepare a second expression vector. Subsequently, a protein conjugate (a+c) comprising anti-mouse PD-1 Fab-Fc (A) and anti-mouse PD-1 Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "c") was obtained using the same method as in Example 2-1 (see Figure 4b).

[0183] 5-2. Purification of mouse PD-1 antibody-IL2 mutant protein complex (a+c)

[0184] The mouse PD-1 antibody-IL2 mutant protein complex (a+c) obtained through the cell line culture prepared in Example 5-1 was purified to obtain a highly pure substance (purity of 98% or more).

[0185] First, the mouse PD-1 antibody-IL2 mutant protein conjugate (a+c) was used. After removing fine residues, primary purification was performed using the same method as in Example 3-2. The filtered medium was then loaded onto a Protein A column stabilized with phosphate buffer saline (pH 7.4). Non-specifically bound proteins were washed away using the same buffer, followed by another wash using a buffer solution containing 0.05 M Citric acid (pH 5.0). Proteins specifically bound to the Protein A column were eluted using a decreasing pH gradient with 0.02 M Citric acid (pH 5.0 and pH 3.5), and the sample was neutralized to pH 7.2 with 1 M Tris.

[0186] Subsequently, to remove impurities derived from substances remaining in the sample separated through the affinity column, a secondary purification was performed using a hydrophobic interaction column (Phenyl HP, Cytiva). Specifically, the sample eluted and neutralized from the Protein-A column was titrated to pH 7.0 with 1M Citric acid and applied to a Phenyl HP column stabilized with 0.02M sodium phosphate (pH 7.0) buffer and 0.8M ammonium sulfate (pH 7.0). Nonspecifically bound proteins were then washed out using the same buffer. Subsequently, mouse PD-1 antibody-IL2 mutant protein complexes (a+c) were isolated by elution using a decreasing gradient of buffer solution containing 0.02M sodium phosphate (pH 7.0). The obtained mouse PD-1 antibody-IL2 mutant protein complex (a+c) was stabilized in phosphate buffer saline (pH 7.4) and then subjected to purity analysis using size exclusion HPLC (TSK-3000SWxL, 7.8 mm x 30 cm, Tosoh).

[0187] FIG. 5A shows the results of purifying a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a Protein-A affinity column purification method.

[0188] Figure 5B shows the results of purifying a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment using a hydrophobic interaction column purification method.

[0189] Figure 5C shows the results of a purity analysis of a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) according to one embodiment.

[0190] As a result, as shown in Figure 5A, proteins that specifically bind to the Protein-A affinity column were confirmed depending on the elution buffer used in the Protein-A affinity column purification. Specifically, mouse PD-1 antibody-IL2 variant 14 protein complexes (a+c) that specifically bind to the column were confirmed in 2800 to 2900 ml of eluate.

[0191] In addition, as shown in Figure 5B, it was confirmed that the remaining impurities and nonspecifically binding proteins in the eluate used in the Protein A affinity column were removed.

[0192] Furthermore, as shown in Figure 5C, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) obtained through all of the above purification processes was confirmed to have a purity of 98.6% with a retention time of 15.639 minutes.

[0193] That is, it can be seen that the mouse PD-1 antibody-IL2 mutant protein complex (a+c) according to one embodiment is purified to a high degree.

[0194] 5-3. Confirmation of receptor binding ability of mouse PD-1 antibody-IL2 mutant protein complex (a+c)

[0195] The receptor binding ability of the mouse PD-1 antibody-IL2 mutant 1 protein complex (a+c) and the mouse PD-1 antibody-IL2 mutant 14 protein complex (a+c) prepared in Example 5-2 was determined in the same manner as in Example 3-3, except that the mouse PD-1 antibody-IL2 mutant 14 protein complex (a+c) was serially diluted in two-fold increments in the concentration range of 1.56 to 8000 nM and developed.

[0196] [Table 8]

[0197] As a result, as shown in Table 8, the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) and the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) showed similar binding affinities to IL2Rβ, 5,610 nM and 6,290 nM, respectively. On the other hand, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) showed binding affinities to IL2Rβγ, 4.51 nM and 35.3 nM, respectively, demonstrating that the binding affinity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) was approximately 7.8-fold lower than that of the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c).

[0198] This means that while the binding ability of the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) and the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) to IL2Rβ was similar, the binding ability of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) to IL2Rγ was reduced by approximately 7.8-fold compared to the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c).

[0199] 5-4. Confirmation of activity of mouse PD-1 antibody-IL2 mutant protein complex (a+c) on human immune cells

[0200] The effect of mouse PD-1 antibody-IL2 variant protein complexes (a+c) on the activation of human immune cells was determined in the same manner as in Examples 3-4, except that the mouse PD-1 antibody-IL2 variant 1 protein complex, mouse PD-1 antibody-IL2 variant 2 protein complex, mouse PD-1 antibody-IL2 variant 3 protein complex, mouse PD-1 antibody-IL2 variant 4 protein complex, mouse PD-1 antibody-IL2 variant 7 protein complex, mouse PD-1 antibody-IL2 variant 8 protein complex, mouse PD-1 antibody-IL2 variant 14 protein complex, and mouse PD-1 antibody-IL2 variant 15 protein complex prepared in Example 5-2 and a mouse PD-1 antibody-IL2 variant comparison group were used.

[0201] [Table 9]

[0202] As a result, as shown in Table 9, in the positive control group (Aldesleukin), the EC of CD8+ T cells / regulatory T cells was 50 The ratio of these values ​​was 21.4, while the mouse PD-1 antibody-IL2 mutant protein conjugate (a+c) prepared in Example 5-2 showed a ratio of 0.5 to 1.2. In other words, it can be seen that the positive control group more strongly activated regulatory T cells, while the mouse PD-1 antibody-IL2 mutant protein conjugate (a+c) according to one embodiment selectively activated CD8+ T cells, which are effector T cells, rather than regulatory T cells. In other words, it can be seen that the IL2 mutant, even in a structure in which it is linked to the C-terminus of the F C region, still retains its inherent property of selectively activating CD8+ T cells.

[0203] Furthermore, the activity of the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c) against Tregs and CD8+ T cells was 11.91±8.02 nM and 9.61±1.44 nM, respectively, while the activity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) against Tregs and CD8+ T cells was 320.7±279.1 nM and 161.1±70.3 nM, respectively, confirming that the activity of the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) against Tregs and CD8+ T cells was reduced compared to the mouse PD-1 antibody-IL2 variant 1 protein complex (a+c). This is due to a reduced binding affinity to IL2Rγ (approximately 7.8-fold), as confirmed in Example 5-3. In addition, the EC of effector T cells / regulatory T cells was also reduced. 50 The ratio of values ​​is 0.5, indicating that effector T cells (CD8+ T cells) are selectively activated compared to regulatory T cells.

[0204] Therefore, the mouse PD-1 antibody-IL2 mutant protein complex (a+c) according to one embodiment can be involved in immune responses related to anti-cancer effects by selectively activating effector T cells over regulatory T cells.

[0205] 5-5. Confirmation of anti-cancer activity of mouse PD-1 antibody-IL2 mutant protein complex (a+c)

[0206] The anti-cancer activity of the mouse PD-1 antibody-IL2 mutant protein complex (a+c) according to one embodiment was confirmed. Specifically, MC38 cells (1.0 × 10 6 After a one-week acclimatization period, C57BL / 6 mice (female, 6 weeks old, Coretec Co., Ltd.) were subcutaneously injected with 1000 cells / mouse into the right flank to prepare a syngeneic mouse model bearing colon cancer. 3When the tumor growth rate reached 100%, the mice were divided into drug treatment groups of 3 to 6 mice each, and the mouse PD-1 antibody-IL2 mutant 14 protein complex (a+c) prepared in Example 5-2 was intraperitoneally administered twice a week at doses of 1, 3, 5, and 10 mg / kg. Vehicle was used as the negative control, and anti-PD1 antibody J43 (clone) 3 mg / kg and J43 3 mg / kg + IL2 comparison group-Fc complex 1.4 mg / kg were used as positive controls. The tumor growth inhibitory effect of drug administration was then observed for up to 19 days after the initial drug administration.

[0207] FIG. 6A shows the results of comparing the anti-cancer activity of a mouse PD-1 antibody-IL2 variant 14 protein conjugate (a+c) according to one embodiment, a negative control group, and a positive control group in a mouse model xenografted with colon cancer.

[0208] Figure 6B shows the results of comparing the weight change in a mouse model xenografted with colon cancer due to the anti-cancer activity of a specific example of a mouse PD-1 antibody-IL2 variant 14 protein complex (a+c), a negative control group, and a positive control group.

[0209] As a result, as shown in Figures 6A and 6B, while the tumor volume increased over time in the negative control group and the positive control group, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) prepared in Example 5-2 demonstrated a dose-dependent cancer cell growth inhibitory effect compared to immediately after administration. In particular, compared to the positive control group and the combined administration group, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) at 5 mg / kg and 10 mg / kg demonstrated a superior cancer cell growth inhibitory effect. In other words, the mouse PD-1 antibody-IL2 variant 14 protein complex (a+c) demonstrated significantly superior anti-cancer activity compared to the combined administration of a PD-1 monoclonal antibody and an IL2 comparison group-Fc protein complex.

[0210] Therefore, the mouse PD-1 antibody-IL2 mutant protein complex (a+c) according to one embodiment specifically activates effective T cells compared to existing immune-mediated anti-cancer therapeutic agents, and may be useful in preventing or treating various immune diseases related to cancer.

[0211] Example 6. Preparation of human PD-1 antibody-IL-2 mutant protein conjugate (A+C)

[0212] 6-1. Cloning and cultivation of human PD-1 antibody-IL2 mutant protein complex (A+C)

[0213] Protein complexes containing a human PD-1 antibody and an IL2 control, IL2 variant 1, and IL2 variant 14 were prepared. Specifically, protein complexes (A+C) containing anti-human PD-1 Fab-Fc (hereinafter referred to as "A") and anti-human PD-1 Fab-Fc-C-terminal IL2 variant (hereinafter referred to as "C") were obtained in the same manner as in Example 5-1, except that a polynucleotide encoding the human PD-1 antibody heavy chain variable region-constant region and a polynucleotide encoding the human PD-1 antibody light chain constant region-light chain variable region were used.

[0214] 6-2. Purification of human PD-1 antibody-IL2 mutant protein complex (A+C)

[0215] The human PD-1 antibody-IL2 mutant protein conjugate (A+C) obtained through the cell line culture prepared in Example 6-1 was purified in the same manner as in Example 5-2 and then stabilized in phosphate buffer saline (pH 7.4) to obtain a highly purified human PD-1 antibody-IL2 mutant protein conjugate (A+C). Purity analysis was then performed in the same manner as in Example 5-2.

[0216] 6-3 Confirmation of binding affinity of human PD-1 antibody-IL2 mutant protein complex (A+C)

[0217] The receptor binding activity of the human PD-1 antibody-IL2 mutant protein conjugate (A+C) was determined in the same manner as in Example 3-3, except that the human PD-1 antibody-IL2 comparison protein conjugate (A+C), human PD-1 antibody-IL2 variant 1 protein conjugate (A+C), and human PD-1 antibody-IL2 variant 14 protein conjugate (A+C) prepared in Example 6-2 were serially diluted in 2-fold increments over the concentration range of 1.56 to 8000 nM and developed. Pembrolizumab was used as the positive control for human PD-1, and human PD-1 antibody-IL2 variant 1 was used as the control. The results are shown in Tables 10 and 11 below.

[0218] [Table 10]

[0219] [Table 11]

[0220] As a result, as shown in Table 10, the huPD-1 antibody-IL2 comparison protein conjugate (A+C), human PD-1 antibody-IL2 variant 1 protein conjugate (A+C), and human PD-1 antibody-IL2 variant 14 protein conjugate (A+C), all prepared in Example 6-2, and the positive control, exhibited binding strengths of 2.25 to 2.95 nM, confirming that all of them exhibited similar levels of binding strength to human PD-1.

[0221] As shown in Table 11, the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) were confirmed to be non-reactive with IL2Rα. On the other hand, the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) showed similar levels of binding affinity to IL2Rβ, with binding affinities of 5.462 nM and 5.611 nM, respectively, and the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) were confirmed to bind to IL2Rβγ with binding affinities of 2.92 nM and 68.3 nM, respectively. These results demonstrate that the binding affinity of the human PD-1 antibody-IL2 variant 14 protein complex (A+C) to IL2Rβ was similar to that of the human PD-1 antibody-IL2 variant 1 protein complex (A+C), but its binding affinity to IL2Rγ was reduced. Specifically, the binding affinity of the human PD-1 antibody-IL2 variant 14 protein complex (A+C) to IL2Rβγ and IL2Rαβγ was approximately 23.4-fold and 32.6-fold reduced, respectively, compared to the human PD-1 antibody-IL2 variant 1. In other words, the human PD-1 antibody-IL2 variant 1 protein complex (A+C) and the human PD-1 antibody-IL2 variant 14 protein complex (A+C) did not bind to ILRα and also exhibited weaker binding to IL2Rγ.

[0222] Therefore, in one embodiment, the human PD-1 antibody-IL2 variant protein conjugate (A+C) is intended to induce higher activation in CD8+ T cells compared to regulatory T cells, and is characterized in that its binding affinity to IL2Rα is abolished and its binding affinity to IL2Rγ is regulated through the substitution of specific amino acids in order to induce selective activation of CD8+ T cells expressing IL2Rβγ.

[0223] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A protein comprising an IL2 mutant, The IL2 variant is a protein comprising glutamic acid (E) at position 35, alanine (A) at position 38, lysine (K) at position 42, and serine (S) at position 125.

2. The IL2 mutant is 2. The protein of claim 1, further comprising one or more amino acids selected from the group consisting of methionine (M), arginine (R), alanine (A), leucine (L), serine (S), phenylalanine (F), valine (V), isoleucine (I), glutamine (Q), tryptophan (W), asparagine (N), and threonine (T) at one or more positions selected from the group consisting of positions 18 and 19.

3. The IL2 mutant is methionine (M) or arginine (R) at position 18, and The protein of claim 2 , which contains serine (S) at position 19.

4. The IL2 mutant is The protein of claim 1, further comprising one or more amino acids at position 126 selected from the group consisting of threonine (T) and isoleucine (I).

5. The IL2 mutant is The protein of claim 1, comprising an amino acid selected from the group consisting of SEQ ID NOs: 1 to 15.

6. The protein of claim 1 , wherein the protein comprises an Fc region attached by a linker or carrier.

7. a protein complex comprising a first polypeptide comprising a first CH3 antibody constant region and a second polypeptide comprising a second CH3 antibody constant region, wherein the first polypeptide and the second polypeptide form a heterodimer; an antibody or antigen-binding fragment thereof against an immune checkpoint, or an antibody or antigen-binding fragment thereof against a tumor-associated antigen, at the N-terminus of at least one of the first polypeptide or the second polypeptide; A protein complex comprising an IL2 mutant at either one or more of the N-terminus and C-terminus of the first polypeptide or the second polypeptide.

8. the first CH3 antibody constant region comprises a tryptophan (W) at position 366, and the second CH3 antibody constant region comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407; The protein complex of claim 7 , comprising glycine (G) or phenylalanine (F) at position 351 of the second CH3 antibody constant region (wherein the position of the amino acid is according to the Kabat EU index).

9. the first CH3 antibody constant region comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407, and the second CH3 antibody constant region comprises a tryptophan (W) at position 366; The protein complex of claim 7 , comprising glycine (G) or phenylalanine (F) at position 351 of the first CH3 antibody constant region (wherein the position of the amino acid is according to the Kabat EU index).

10. the first CH3 antibody constant region comprises a tryptophan (W) at position 366, and the second CH3 antibody constant region comprises a glycine (G) at position 351, a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407; The protein complex of claim 7 , which comprises phenylalanine (F) or tryptophan (W) at position 351 of the first CH3 antibody constant region (wherein the position of the amino acid is according to the Kabat EU index).

11. the first CH3 antibody constant region comprises glycine (G) at position 351, serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407; and the second CH3 antibody constant region comprises tryptophan (W) at position 366; The protein complex of claim 7 , which comprises phenylalanine (F) or tryptophan (W) at position 351 of the second CH3 antibody constant region (wherein the position of the amino acid is according to the Kabat EU index).

12. A protein complex as described in claim 7, wherein the first CH antibody constant region and the second CH antibody constant region contain alanine (A) at positions 234 and 235.

13. The protein complex of claim 7, wherein the immune checkpoint is any one or more selected from the group consisting of PD-L1, PD-1, LAG-3, VISTA, BTLA, TIM-3, TIGIT, and CTLA-4.

14. The protein complex of claim 7, wherein the tumor-specific antigen is any one or more selected from the group consisting of PD-L1, EGFR, HER-2, B7H3, GPC3, CEA, TROP, and PSMA.

15. The protein complex of claim 7 , wherein the second polypeptide and the IL2 variant are linked by one or more linkers or carriers.

16. The protein complex of claim 15, wherein the linker comprises the amino acid sequence of SEQ ID NO:

18.

17. A polynucleotide encoding a protein or protein complex according to any one of claims 1 to 16.

18. 17. A method for producing a protein or protein complex, comprising transforming a cell with an expression vector encoding the protein or protein complex of any one of claims 1 to 16.

19. A protein complex containing an IL2 mutant, produced by the method of claim 18.

20. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the protein or protein complex according to any one of claims 1 to 16.

21. 20. A method for preventing or treating cancer, comprising administering to an individual in need thereof a protein or protein complex according to any one of claims 1 to 16.

22. 20. Use of a protein or protein complex according to any one of claims 1 to 16 for the manufacture of a cancer therapeutic agent.

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